An exon-biased biophysical approach and NMR spectroscopy define the secondary structure of a conserved helical element within the HOTAIR long non-coding RNA

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Highlights

  • -Structure determination is challenging for lncRNAs that contain multiple splice isoforms.

  • -An exon-biased biophysical approach that includes NMR and chemical probing was developed to identify homogenous conformations within the exons of HOTAIR.

  • -HOTAIR exon 4 contains a well-defined helical conformation, previously identified as a conserved RNA structure.

  • -Combined NMR with chemical probing methods provide accuracy in the secondary structure determination of large RNA molecules.

Abstract

HOTAIR is a large, multi-exon spliced non-coding RNA proposed to function as a molecular scaffold and competes with chromatin to bind to histone modification enzymes. Previous sequence analysis and biochemical experiments identified potential conserved regions and characterized the full length HOTAIR secondary structure. Here, we examine the thermodynamic folding properties and structural propensity of the individual exonic regions of HOTAIR using an array of biophysical methods and NMR spectroscopy. We demonstrate that different exons of HOTAIR contain variable degrees of heterogeneity, and identify one exonic region, exon 4, that adopts a stable and compact fold under low magnesium concentrations. Close agreement of NMR spectroscopy and chemical probing unambiguously confirm conserved base pair interactions within the structural element, termed helix 10 of exon 4, located within domain I of human HOTAIR. This combined exon-biased and integrated biophysical approach introduces a new strategy to examine conformational heterogeneity in lncRNAs and emphasizes NMR as a key method to validate base pair interactions and corroborate large RNA secondary structures.

Introduction

Long noncoding RNA (lncRNA) are dynamic modulators of gene expression and can function as molecular scaffolds, associating with chromatin modifying complexes near genomic loci to influence chromatin structure and gene expression (Spitale et al., 2011, Rinn and Chang, 2012, Marchese et al., 2017). In terms of gene architecture and sequence similarity, most lncRNAs do not have identifiable homologs but there are thousands of human lncRNAs that have defined homologs and share similar expression levels in vertebrate genomes. Comparative transcriptome studies show that some lncRNAs exhibit a varying degree of sequence conservation across short nucleotide stretches that reside within exonic regions, suggesting that conserved functions require only short sequence or structural regions that can be tolerated within the syntenic architecture (Hezroni et al., 2015, Quinn et al., 2016, Ulitsky, 2016).

In addition, many homologous lncRNAs are alternatively spliced at levels approaching that of mRNAs and are thought to have rapidly evolved to acquire a functional importance (Schorderet and Duboule, 2011, Haerty and Ponting, 2015, Ulitsky, 2016). During evolution, alternative splicing and exon number globally increase, while exon length decreases (Koralewski and Krutovsky, 2011, Haerty and Ponting, 2015, Lin et al., 2016). This notion is also illustrated in the analysis of tertiary-quaternary structure in protein coding genes, where protein domain boundaries can correlate with exon boundaries (Richardson, 1981, Liu and Grigoriev, 2004). These generalities suggest that exon boundaries can stabilize or destabilize RNA elements and demarcate regions within large RNAs that contain a defined tertiary fold.

We sought to explore the correlation of gene sequence elements and domain boundaries within the context of lncRNAs and developed an RNA secondary structure determination strategy termed Exon-Biased structure probing. This method assumes that a secondary structure fold can be contained within an exonic region of the lncRNA. As an example, we analyzed the heterogeneity and folding properties of individual exons of the human HOX transcript antisense RNA (hHOTAIR) using native gel electrophoresis, thermal melting, and analytical ultracentrifugation. In addition, we interrogated the most stable and homogenously folded hHOTAIR exon in vitro using chemical probing and NMR spectroscopy.

HOTAIR is a classic example of a lncRNA involved in silencing specific homeotic genes in embryonic stem cells and whose overexpression is associated with tumor metastasis and poor prognosis (Rinn et al., 2007, Gupta et al., 2010). Human HOTAIR primarily contains six exons and was identified to function in trans, influencing the transcriptional repression of a distant chromosomal domain (Rinn et al., 2007, Gupta et al., 2010, Tsai et al., 2010). Based upon the UCSC genome browser, targeted RNA-sequencing of hHOTAIR has revealed the existence of at least six isoforms, with alternative splicing events generating an additional 16 different isoforms (Kent et al., 2002). Although the physiological regulation of hHOTAIR alternative splicing is unknown, it is possible that different splice isoforms could impact the tertiary fold of the RNA, potential RNA-protein interactions, and the extent of transcriptional repression.

We selected hHOTAIR using an exon-biased mapping approach because its RNA secondary structure has been extensively studied in vitro (Kertesz et al., 2010, He et al., 2011, Wu et al., 2013, Somarowthu et al., 2015, Portoso et al., 2017, Spokoini-Stern et al., 2020). The full-length hHOTAIR has been defined using multiple chemical probing strategies, identifying four large domains (Domain I-IV) that contain specific structured regions that are proposed to contain high sequence co-variation (Somarowthu et al., 2015). Domain I has the highest degree of covariation support when compared to other regions of hHOTAIR, yet some R-scape and power covariation studies suggest that hHOTAIR does not contain any evolutionarily conserved RNA structures (Rivas et al., 2017, Rivas et al., 2020). In this paper, we show that at least one region of hHOTAIR can adopt a homogenous tertiary fold and represents a structural domain that is preserved within an exonic boundary of hHOTAIR, suggestive of an evolutionarily conserved RNA structure. In addition, we propose that this exon-focused biophysical approach, when combined with hybrid structural bioinformatic studies, may serve as a generalizable strategy to examine the evolution of conserved lncRNA secondary structure within mammalian genomes.

Section snippets

HOTAIR RNA synthesis and purification

Double stranded (ds) DNA templates for each HOTAIR exon were generated from the pLZRS-HOTAIR plasmid (H. Chang, Addgene Plasmid ID #26110) (Gupta et al., 2010) by polymerase chain-reaction (PCR) with primer pairs as listed in Table S1. Forward primers contain a T7 RNA Polymerase binding site to initiate in vitro transcription reactions (Milligan et al., 1987). PCR reactions were purified via spin-column (Qiagen), eluted in RNAse-free water, and quantified by UV absorbance at 260 nm.

In vitro

Results

To probe the general interplay between splicing and the folding properties of lncRNAs, we sought to characterize the conformational heterogeneity of individual, isolated hHOTAIR exonic transcripts and identify potential regions that contain a highly stable secondary structure. HOTAIR exons 1 (60 nt) and 2 (126 nt) displayed significant heterogeneity, regardless of treatment (Fig. 1). In particular, exon 1 exhibited a marked propensity to dimerize in both non-denatured (‘native’) and refolded

Discussion

The characterization of functionally relevant lncRNA-mediated biological mechanisms requires multiple experimental biophysical and bioinformatic approaches that probe the structure and thermodynamic properties of the RNA target (Butcher and Pyle, 2011, Pyle, 2014, Chu et al., 2015, Yao et al., 2017, Jones and Sattler, 2019). Although there still remains limited and conflicting mechanistic insight into the structure–function relationships of HOTAIR (Tsai et al., 2010, Wu et al., 2013, Somarowthu

Conclusions

We combined an exon-biased method with chemical probing and NMR to determine that HOTAIR can form independent structural domains with varying degrees of heterogeneity and thermodynamic stability. Although this method may be applied to large ncRNAs with defined splice boundaries, the exon-biased structure determination of HOTAIR with NMR spectroscopy and chemical probing does validate that the conserved helix 10 serves as a core helical element within the 5′ region of HOTAIR (Somarowthu et al.,

CRediT authorship contribution statement

Ainur Abzhanova: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Alexander Hirschi: Conceptualization, Data curation, Formal analysis, Writing - original draft. Nicholas J. Reiter: Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing - original draft, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We would like to acknowledge the laboratory of Dr. Michael P. Stone for assistance with thermal melting experiments, Dr. Markus Voehler and the Vanderbilt University Center for Structural Biology, and Dr. Marco Tonelli and the National Magnetic Resonance Facility at Madison (NMRFAM). We thank Dr. William J Martin for comments and the laboratories of Drs. Manuel Ascano and Gregor Neuert (Vanderbilt University Medical Center) for discussions.

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    Authors contributed equally to this work.

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    Present address: Mammoth Biosciences, 279 E Grand Ave., South San Francisco, CA 94080, United States.

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